METHODS FOR TAGGING AND ENCODING OF PRE-EXISTING COMPOUND LIBRARIES
20220275362 · 2022-09-01
Assignee
Inventors
Cpc classification
C12N15/1065
CHEMISTRY; METALLURGY
C40B50/04
CHEMISTRY; METALLURGY
C12N15/1068
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to methods of encoding pre-existing compounds with oligonucleotide tags. In particular, libraries of pre-existing compounds are tagged with oligonucleotides in order to encode identifying information, thereby improving methods of screening and identifying compounds having a desired property.
Claims
1. A method of producing an encoded chemical entity, the method comprising: (a) reacting a chemical entity with a bifunctional linker, the bifunctional linker comprising a carbene precursor group and a first cross-linking group, under conditions sufficient to produce a first conjugate comprising the chemical entity and the first cross-linking group; (b) reacting the first conjugate with a second conjugate, the second conjugate comprising an oligonucleotide headpiece and a second cross-linking group, under conditions sufficient to produce a third conjugate comprising the chemical entity and the oligonucleotide headpiece; and (c) ligating a first oligonucleotide tag to the oligonucleotide headpiece of the third conjugate, thereby producing an encoded chemical entity.
2. The method of claim 1, wherein the bifunctional linker is volatile.
3. The method of claim 1 or 2, wherein the bifunctional linker has the structure:
A-L.sup.1-R.sup.1 Formula I wherein A is the carbene precursor group; L.sup.1 is a linker; and R.sup.1 is the first cross-linking group.
4. The method of any one of claims 1 to 3, wherein the carbene precursor group is a photo-reactive carbene precursor group.
5. The method of claim 4, wherein the photo-reactive carbene precursor group is a diazirine.
6. The method of any one of claims 1 to 5, wherein the carbene precursor group comprises the structure: ##STR00016##
7. The method of any one of claims 3 to 6, wherein L.sup.1 is C.sub.1-C.sub.6 alkylene.
8. The method of claim 7, wherein L.sup.1 is C.sub.2 alkylene.
9. The method of any one of claims 1 to 8, wherein the first cross-linking group is a sulfhydryl-reactive cross-linking group, an amino-reactive cross-linking group, a carboxyl-reactive cross-linking group, a carbonyl-reactive cross-linking group, or a triazole-forming cross-linking group.
10. The method of claim 9, wherein the first cross-linking group is a triazole-forming cross-linking group.
11. The method of any one of claims 1 to 10, wherein the first cross-linking group is an azide.
12. The method of any one of claims 1 to 11, wherein the bifunctional linker has the structure: ##STR00017##
13. The method of any one of claims 1 to 12, wherein the second conjugate has the structure:
B-L.sup.2-R.sup.2 Formula II wherein B is the oligonucleotide headpiece; L.sup.2 is a linker; and R.sup.2 is the second cross-linking group.
14. The method of any one of claims 1 to 13, wherein the oligonucleotide headpiece comprises a hairpin structure.
15. The method of claim 13 or 14, wherein the second cross-linking group is a sulfhydryl-reactive cross-linking group, an amino-reactive cross-linking group, a carboxyl-reactive cross-linking group, a carbonyl-reactive cross-linking group, or a triazole-forming cross-linking group.
16. The method of claim 15, wherein the second cross-linking group is a triazole-forming cross-linking group.
17. The method of claim 16, wherein the second cross-linking group comprises a dibenzocyclooctyne group.
18. The method of claim 17, wherein the second cross-linking group comprises the structure: ##STR00018##
19. The method of any one of claims 1 to 18, wherein the method further comprises producing the second conjugate by reacting a fourth conjugate comprising an oligonucleotide headpiece and a cross-linking group with a fifth conjugate of Formula III:
R.sup.3-L.sup.3-R.sup.4 Formula III wherein R.sup.3 and R.sup.4 are, independently, cross-linking groups; and L.sup.3 is a linker, under conditions sufficient to produce the second conjugate.
20. The method of claim 19, wherein R.sup.3 is a triazole-forming cross-linking group.
21. The method of claim 20, wherein R.sup.3 comprises a dibenzocyclooctyne group.
22. The method of claim 20, wherein R.sup.3 comprises the structure: ##STR00019##
23. The method of any one of claims 19 to 22, wherein R.sup.4 is a sulfhydryl-reactive cross-linking group, an amino-reactive cross-linking group, a carboxyl-reactive cross-linking group, a carbonyl-reactive cross-linking group, or a triazole-forming cross-linking group.
24. The method of claim 23, wherein R.sup.4 is an amino-reactive cross-linking group.
25. The method of claim 24, wherein R.sup.4 comprises a N-hydroxysuccinimide group.
26. The method of any one of claims 19 to 25, wherein the second conjugate has the structure:
B-L.sup.4-R.sup.5 Formula IV wherein B is the oligonucleotide headpiece; L.sup.4 is a linker; and R.sup.5 is the second cross-linking group.
27. The method of claim 26, wherein the second cross-linking group is an amino group.
28. The method of any one of claims 1 to 27, wherein the method further comprises, prior to step (c), ligating a headpiece extension sequence to the headpiece.
29. The method of any one of claims 1 to 28, wherein the method further comprises ligating one or more further tags to the encoded chemical entity after step (c).
30. The method of claim 29, wherein the method further comprises ligating at least three further tags to the encoded chemical entity after step (c).
31. The method of claim 30, wherein the method comprises one-pot ligation.
32. The method of claim 31, wherein the one-pot ligation comprises the ligation of the headpiece extension sequence to the headpiece and the ligation of the at least three further tags to the encoded chemical entity.
33. The method of any one of claims 29 to 32, wherein the first oligonucleotide tag and the one or more further tags comprise orthogonal overlap architectures.
34. The method of any one of claims 1 to 33, wherein the method further comprises ligating a tailpiece to the encoded chemical entity.
35. The method of any one of claims 1 to 34, wherein the chemical entity does not comprise an N—H or O—H bond.
36. The method of any one of claims 1 to 35, wherein the conditions of step (b) do not comprise a metal catalyst.
37. The method of any one of claims 1 to 36, wherein the method further comprises purifying the encoded chemical entity after step (c).
38. The method of claim 37, wherein the purifying comprises high performance liquid chromatography (HPLC).
39. The method of any one of claims 1 to 38, wherein the conditions of step (a) comprises irradiation.
40. A library comprising a plurality of encoded chemical entities produced by the method of any one of claims 1 to 39.
41. The library of claim 40, wherein the plurality of encoded chemical entities is not physically separated.
42. The library of claim 40 or 41, wherein the plurality of encoded chemical entities comprises at least 1,000,000 different chemical entities.
43. The library of any one of claims 40 to 42, wherein the plurality of encoded chemical entities comprises at least 5,000,000 different chemical entities.
44. The library of any one of claims 40 to 43, wherein the plurality of encoded chemical entities comprises at least 10,000,000 different chemical entities.
45. The library of claim 40 or 41, wherein the plurality of encoded chemical entities comprises about 1,000,000 to about 5,000,000 different chemical entities.
46. The library of claim 40 or 41, wherein the plurality of encoded chemical entities comprises about 5,000,000 to about 10,000,000 different chemical entities.
47. A method of screening a plurality of chemical entities, the method comprising: (a) contacting a target with an encoded chemical entity prepared by a method of any one of claims 1 to 39 and/or a library of any one of claims 40 to 46; and (b) selecting one or more encoded chemical entities having a predetermined characteristic for the target, as compared to a control, thereby screening a plurality of the chemical entities.
48. The method of claim 47, where the predetermined characteristic comprises increased binding for the target, as compared to a control.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0146] The disclosure features a method of tagging large libraries of pre-existing compounds, e.g., libraries containing millions of individual compounds, with oligonucleotide tags in order to encode each member of the libraries with identifying information. The resulting encoded libraries can then be screened against targets (e.g., therapeutic targets such as proteins) as a mixture of the individual encoded compounds. This enables a robust and rapid method for identifying compounds of interest (e.g., drug leads, drug candidates, and/or tool compounds).
Encoded Chemical Entities
[0147] This invention features encoded chemical entities including chemical entities (e.g., pre-existing chemical entities), bifunctional linkers, one or more oligonucleotide tags, and headpieces operatively associated with (i) the chemical entities via the bifunctional linkers; and (ii) the one or more oligonucleotide tags. Libraries of encoded chemical entities including chemical entities, bifunctional linkers, one or more oligonucleotide tags, and headpieces are further described below.
Chemical Entities
[0148] The libraries of pre-existing chemical entities (e.g., compounds) or members can include one or more unique compounds.
Bifunctional Linkers
[0149] The bifunctional linker between the headpiece and a chemical entity can be varied to provide an appropriate linking moiety and/or to increase the solubility of the headpiece in organic solvent. A wide variety of linkers are commercially available that can couple the headpiece with the small molecule library. The bifunctional linker typically consists of linear or branched chains and may include a C.sub.1-10 alkyl, a heteroalkyl of 1 to 10 atoms, a C.sub.2-10 alkenyl, a C.sub.2-10 alkynyl, C.sub.5-10 aryl, a cyclic or polycyclic system of 3 to 20 atoms, a phosphodiester, a peptide, an oligosaccharide, an oligonucleotide, an oligomer, a polymer, or a poly alkyl glycol (e.g., a poly ethylene glycol, such as —(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2—, where n is an integer from 1 to 50), or combinations thereof.
[0150] The bifunctional linker may provide an appropriate linking moiety between the headpiece and a chemical entity of the library. In certain embodiments, the bifunctional linker includes three parts. Part 1 may be a reactive group, which forms a covalent bond with DNA, such as, e.g., a carboxylic acid, preferably activated by a N-hydroxy succinimide (NHS) ester to react with an amino group on the DNA (e.g., amino-modified dT), an amidite to modify the 5′ or 3′-terminus of a single-stranded headpiece (achieved by means of standard oligonucleotide chemistry), chemical-reactive pairs (e.g., azido-alkyne cycloaddition optionally in the presence of Cu(I) catalyst, or any described herein), or thiol reactive groups. Part 2 may also be a reactive group, which forms a covalent bond with the chemical entity, either building block A.sub.n or a scaffold. Such a reactive group are, e.g., an amine, a thiol, an azide, or an alkyne. Part 3 may be a chemically inert linking moiety of variable length, introduced between Part 1 and 2. Such a linking moiety can be a chain of ethylene glycol units (e.g., PEGs of different lengths), an alkane, an alkene, a polyene chain, or a peptide chain. The linker can contain branches or inserts with hydrophobic moieties (such as, e.g., benzene rings) to improve solubility of the headpiece in organic solvents, as well as fluorescent moieties (e.g. fluorescein or Cy-3) used for library detection purposes. Hydrophobic residues in the headpiece design may be varied with the linker design to facilitate library synthesis in organic solvents. For example, the headpiece and linker combination is designed to have appropriate residues wherein the octanol:water coefficient (P.sub.oct) is from, e.g., 1.0 to 2.5.
[0151] Linkers can be empirically selected for a given small molecule library design, such that the library can be synthesized in organic solvent, for example, in 15%, 25%, 30%, 50%, 75%, 90%, 95%, 98%, 99%, or 100% organic solvent. The linker can be varied using model reactions prior to library synthesis to select the appropriate chain length that solubilizes the headpiece in an organic solvent. Exemplary linkers include those having increased alkyl chain length, increased polyethylene glycol units, branched species with positive charges (to neutralize the negative phosphate charges on the headpiece), or increased amounts of hydrophobicity (for example, addition of benzene ring structures).
[0152] Linkers may also be branched, where branched linkers are well known in the art and examples can consist of symmetric or asymmetric doublers or a symmetric trebler. See, for example, Newcome et al., Dendritic Molecules: Concepts, Synthesis, Perspectives, VCH Publishers (1996); Boussif et al., Proc. Natl. Acad. Sci. USA 92:7297-7301 (1995); and Jansen et al., Science 266:1226 (1994).
[0153] Linkers optionally include one or more cross-linking groups. Examples of cross-linking groups include azide, carbene precursor group, and alkyne.
Cross-Linking Groups
[0154] A cross-linking group refers to a group comprising a reactive functional group capable of chemically attaching to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. Examples of cross-linking groups include sulfhydryl-reactive cross-linking groups (e.g., groups comprising maleimides, haloacetyls, pyridyldisulfides, thiosulfonates, or vinylsulfones), amine-reactive cross-linking groups (e.g., groups comprising esters such as NHS esters, imidoesters, and pentafluorophenyl esters, or hydroxymethylphosphine), carboxyl-reactive cross-linking groups (e.g., groups comprising primary or secondary amines, alcohols, or thiols), carbonyl-reactive cross-linking groups (e.g., groups comprising hydrazides or alkoxyamines), triazole-forming cross-linking groups (e.g., groups comprising azides or alkynes) or carbene-generating groups such as aziridines.
[0155] Examples of chemically reactive functional groups which may react with cross-linking groups include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.
[0156] Examples of moieties which are sulfhydryl-reactive include α-haloacetyl compounds of the type XCH.sub.2CO— (where X=Br, Cl, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.
[0157] Examples of reactive moieties which are amino-reactive include, for example, alkylating and acylating agents. Representative alkylating agents include:
[0158] (i) α-haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH.sub.2CO— (where X=Br, Cl, or I), for example, as described by Wong Biochemistry 24:5337 (1979);
[0159] (ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);
[0160] (iii) aryl halides such as reactive nitrohaloaromatic compounds;
[0161] (iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988);
[0162] (v) aldehydes and ketones capable of Schiff's base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;
[0163] (vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;
[0164] (vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;
[0165] (viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening;
[0166] (ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and
[0167] (x) α-haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).
[0168] Representative amino-reactive acylating agents include:
[0169] (i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;
[0170] (ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1964);
[0171] (iii) acid halides;
[0172] (iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters;
[0173] (v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;
[0174] (vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984;
[0175] (vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974);
[0176] (viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962); and
[0177] (ix) haloheteroaryl groups such as halopyridine or halopyrimidine.
[0178] Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxyamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).
[0179] Examples of reactive moieties which are “carboxyl-reactive” include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.
[0180] Exemplary cross-linking groups include 2′-pyridyldisulfide, 4′-pyridyldisulfide iodoacetyl, maleimide, thioesters, alkyldisulfides, alkylamine disulfides, nitrobenzoic acid disulfide, anhydrides, NHS esters, aldehydes, alkyl chlorides, alkynes, and azides.
Headpiece
[0181] In the library, the headpiece operatively links each chemical entity to its encoding oligonucleotide tag. Generally, the headpiece is a starting oligonucleotide having two functional groups that can be further derivatized, where the first functional group operatively links the chemical entity (or a component thereof) to the headpiece and the second functional group operatively links one or more tags to the headpiece. A bifunctional linker can optionally be used as a linking moiety between the headpiece and the chemical entity.
[0182] The functional groups of the headpiece can be used to form a covalent bond with a component of the chemical entity and another covalent bond with a tag. The component can be any part of the small molecule, such as a scaffold having diversity nodes or a building block. Alternatively, the headpiece can be derivatized to provide a linker (i.e., a linking moiety separating the headpiece from the small molecule to be formed in the library) terminating in a functional group (e.g., a hydroxyl, amine, carboxyl, sulfhydryl, alkynyl, azido, or phosphate group), which is used to form the covalent linkage with a component of the chemical entity. The linker can be attached to the 5′-terminus, at one of the internal positions, or to the 3′-terminus of the headpiece. When the linker is attached to one of the internal positions, the linker can be operatively linked to a derivatized base (e.g., the C5 position of uridine) or placed internally within the oligonucleotide using standard techniques known in the art. Exemplary linkers are described herein.
[0183] The headpiece can have any useful structure. The headpiece can be, e.g., 1 to 100 nucleotides in length, preferably 5 to 20 nucleotides in length, and most preferably 5 to 15 nucleotides in length. The headpiece can be single-stranded or double-stranded and can consist of natural or modified nucleotides, as described herein. For example, the chemical moiety can be operatively linked to the 3′-terminus or 5′-terminus of the headpiece. In particular embodiments, the headpiece includes a hairpin structure formed by complementary bases within the sequence. For example, the chemical moiety can be operatively linked to the internal position, the 3′-terminus, or the 5′-terminus of the headpiece.
[0184] Generally, the headpiece includes a non-self-complementary sequence on the 5′- or 3′-terminus that allows for binding an oligonucleotide tag by polymerization, enzymatic ligation, or chemical reaction. The headpiece can allow for ligation of oligonucleotide tags and optional purification and phosphorylation steps. After the addition of the last tag, an additional adapter sequence can be added to the 5′-terminus of the last tag. Exemplary adapter sequences include a primer-binding sequence or a sequence having a label (e.g., biotin). In cases where many building blocks and corresponding tags are used (e.g., 100), a mix-and-split strategy may be employed during the oligonucleotide synthesis step to create the necessary number of tags. Such mix-and-split strategies for DNA synthesis are known in the art. The resultant library members can be amplified by PCR following selection for binding entities versus a target(s) of interest.
[0185] The oligonucleotide headpiece of the encoded chemical entity can optionally include one or more primer-binding sequences. For example, the headpiece has a sequence in the loop region of the hairpin that serves as a primer-binding region for amplification, where the primer-binding region has a higher melting temperature for its complementary primer (e.g., which can include flanking identifier regions) than for a sequence in the headpiece. In other embodiments, the encoded chemical entity includes two primer-binding sequences (e.g., to enable PCR) on either side of one or more tags that encode one or more building blocks. Alternatively, the headpiece may contain one primer-binding sequence on the 5′- or 3′-terminus. In other embodiments, the headpiece is a hairpin, and the loop region forms a primer-binding site or the primer-binding site is introduced through hybridization of an oligonucleotide to the headpiece on the 3′ side of the loop. A primer oligonucleotide, containing a region homologous to the 3′-terminus of the headpiece and carrying a primer-binding region on its 5′-terminus (e.g., to enable a PCR reaction) may be hybridized to the headpiece and may contain a tag that encodes a building block or the addition of a building block. The primer oligonucleotide may contain additional information, such as a region of randomized nucleotides, e.g., 2 to 16 nucleotides in length, which is included for bioinformatics analysis.
[0186] The headpiece can optionally include a hairpin structure, where this structure can be achieved by any useful method. For example, the headpiece can include complementary bases that form intermolecular base pairing partners, such as by Watson-Crick DNA base pairing (e.g., adenine-thymine and guanine-cytosine) and/or by wobble base pairing (e.g., guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine). In another example, the headpiece can include modified or substituted nucleotides that can form higher affinity duplex formations compared to unmodified nucleotides, such modified or substituted nucleotides being known in the art.
[0187] The oligonucleotide headpiece of the encoded chemical entity can optionally include one or more labels that allow for detection. For example, the headpiece, one or more oligonucleotide tags, and/or one or more primer sequences can include an isotope, a radioimaging agent, a marker, a tracer, a fluorescent label (e.g., rhodamine or fluorescein), a chemiluminescent label, a quantum dot, and a reporter molecule (e.g., biotin or a his-tag).
[0188] In other embodiments, the headpiece or tag may be modified to support solubility in semi-, reduced-, or non-aqueous (e.g., organic) conditions. Nucleotide bases of the headpiece or tag can be rendered more hydrophobic by modifying, for example, the C5 positions of T or C bases with aliphatic chains without significantly disrupting their ability to hydrogen bond to their complementary bases. Exemplary modified or substituted nucleotides are 5′-dimethoxytrityl-N4-diisobutylaminomethylidene-5-(1-propynyl)-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5′-dimethoxytrityl-5-(1-propynyl)-2′-deoxyuridine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5′-dimethoxytrityl-5-fluoro-2′-deoxyuridine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and 5′-dimethoxytrityl-5-(pyren-1-yl-ethynyl)-2′-deoxyuridine, or 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
[0189] In addition, the headpiece oligonucleotide can be interspersed with modifications that promote solubility in organic solvents. For example, azobenzene phosphoramidite can introduce a hydrophobic moiety into the headpiece design. Such insertions of hydrophobic amidites into the headpiece can occur anywhere in the molecule. However, the insertion cannot interfere with subsequent tagging using additional DNA tags during the library synthesis or ensuing PCR once a selection is complete or microarray analysis, if used for tag deconvolution. Such additions to the headpiece design described herein would render the headpiece soluble in, for example, 15%, 25%, 30%, 50%, 75%, 90%, 95%, 98%, 99%, or 100% organic solvent. Thus, addition of hydrophobic residues into the headpiece design allows for improved solubility in semi- or non-aqueous (e.g., organic) conditions, while rendering the headpiece competent for oligonucleotide tagging. Furthermore, DNA tags that are subsequently introduced into the library can also be modified at the C5 position of T or C bases such that they also render the library more hydrophobic and soluble in organic solvents for subsequent steps of library synthesis.
[0190] In particular embodiments, the headpiece and the first tag can be the same entity, i.e., a plurality of headpiece-tag entities can be constructed that all share common parts (e.g., a primer-binding region) and all differ in another part (e.g., encoding region). These may be utilized in the “split” step and pooled after the event they are encoding has occurred.
[0191] In particular embodiments, the headpiece can encode information, e.g., by including a sequence that encodes the first split(s) step or a sequence that encodes the identity of the library, such as by using a particular sequence related to a specific library.
Oligonucleotide Tags
[0192] The oligonucleotide tags described herein (e.g., a tag or a portion of a headpiece or a portion of a tailpiece) can be used to encode any useful information, such as a molecule, a portion of a chemical entity, the addition of a component (e.g., a scaffold or a building block), a headpiece in the library, the identity of the library, the use of one or more library members (e.g., use of the members in an aliquot of a library), and/or the origin of a library member (e.g., by use of an origin sequence).
[0193] Any sequence in an oligonucleotide can be used to encode any information. Thus, one oligonucleotide sequence can serve more than one purpose, such as to encode two or more types of information or to provide a starting oligonucleotide that also encodes for one or more types of information.
[0194] For example, the first tag can encode for the addition of a first building block, as well as for the identification of the library. In another example, a headpiece can be used to provide a starting oligonucleotide that operatively links a chemical entity to a tag, where the headpiece additionally includes a sequence that encodes for the identity of the library (i.e., the library-identifying sequence). Accordingly, any of the information described herein can be encoded in separate oligonucleotide tags or can be combined and encoded in the same oligonucleotide sequence (e.g., an oligonucleotide tag, such as a tag, or a headpiece).
[0195] A building block sequence encodes for the identity of a building block and/or the type of binding reaction conducted with a building block. This building block sequence is included in a tag, where the tag can optionally include one or more types of sequence described below (e.g., a library-identifying sequence, a use sequence, and/or an origin sequence).
[0196] A library-identifying sequence encodes for the identity of a particular library. In order to permit mixing of two or more libraries, a library member may contain one or more library-identifying sequences, such as in a library-identifying tag (i.e., an oligonucleotide including a library-identifying sequence), in a ligated tag, in a part of the headpiece sequence, or in a tailpiece sequence. These library-identifying sequences can be used to deduce encoding relationships, where the sequence of the tag is translated and correlated with chemical (synthesis) history information. Accordingly, these library-identifying sequences permit the mixing of two or more libraries together for selection, amplification, purification, sequencing, etc.
[0197] A use sequence encodes the history (i.e., use) of one or more library members in an individual aliquot of a library. For example, separate aliquots may be treated with different reaction conditions, building blocks, and/or selection steps. In particular, this sequence may be used to identify such aliquots and deduce their history (use) and thereby permit the mixing together of aliquots of the same library with different histories (uses) (e.g., distinct selection experiments) for the purposes of the mixing together of samples together for selection, amplification, purification, sequencing, etc. These use sequences can be included in a headpiece, a tailpiece, a tag, a use tag (i.e., an oligonucleotide including a use sequence), or any other tag described herein (e.g., a library-identifying tag or an origin tag).
[0198] An origin sequence is a degenerate (random, stochastically-generated) oligonucleotide sequence of any useful length (e.g., about six oligonucleotides) that encodes for the origin of the library member. This sequence serves to stochastically subdivide library members that are otherwise identical in all respects into entities distinguishable by sequence information, such that observations of amplification products derived from unique progenitor templates (e.g., selected library members) can be distinguished from observations of multiple amplification products derived from the same progenitor template (e.g., a selected library member). For example, after library formation and prior to the selection step, each library member can include a different origin sequence, such as in an origin tag. After selection, selected library members can be amplified to produce amplification products, and the portion of the library member expected to include the origin sequence (e.g., in the origin tag) can be observed and compared with the origin sequence in each of the other library members. As the origin sequences are degenerate, each amplification product of each library member should have a different origin sequence. However, an observation of the same origin sequence in the amplification product could indicate multiple amplicons derived from the same template molecule. When it is desired to determine the statistics and demographics of the population of encoding tags prior to amplification, as opposed to post-amplification, the origin tag may be used. These origin sequences can be included in a headpiece, a tailpiece, a tag, an origin tag (i.e., an oligonucleotide including an origin sequence), or any other tag described herein (e.g., a library-identifying tag or a use tag).
[0199] Any of the types of sequences described herein can be included in the headpiece. For example, the headpiece can include one or more of a building block sequence, a library-identifying sequence, a use sequence, or an origin sequence.
[0200] Any of these sequences described herein can be included in a tailpiece. For example, the tailpiece can include one or more of a library-identifying sequence, a use sequence, or an origin sequence.
[0201] Any of tags described herein can include a connector at or in proximity to the 5′- or 3′-terminus having a fixed sequence. Connectors facilitate the formation of linkages (e.g., chemical linkages) by providing a reactive group (e.g., a chemical-reactive group or a photo-reactive group) or by providing a site for an agent that allows for a linkage (e.g., an agent of an intercalating moiety or a reversible reactive group in the connector(s) or cross-linking oligonucleotide). Each 5′-connector may be the same or different, and each 3′-connector may be the same or different. In an exemplary, non-limiting conjugate or encoded chemical entity having more than one tags, each tag can include a 5′-connector and a 3′-connector, where each 5′-connector has the same sequence and each 3′-connector has the same sequence (e.g., where the sequence of the 5′-connector can be the same or different from the sequence of the 3′-connector). The connector provides a sequence that can be used for one or more linkages. To allow for binding of a relay primer or for hybridizing a cross-linking oligonucleotide, the connector can include one or more functional groups allowing for a linkage (e.g., a linkage for which a polymerase has reduced ability to read or translocate through, such as a chemical linkage).
[0202] These sequences can include any modification described herein for oligonucleotides, such as one or more modifications that promote solubility in organic solvents (e.g., any described herein, such as for the headpiece), that provide an analog of the natural phosphodiester linkage (e.g., a phosphorothioate analog), or that provide one or more non-natural oligonucleotides (e.g., 2′-substituted nucleotides, such as 2′-O-methylated nucleotides and 2′-fluoro nucleotides, or any described herein).
[0203] These sequences can include any characteristics described herein for oligonucleotides. For example, these sequences can be included in tag that is less than 20 nucleotides (e.g., as described herein). In other examples, the tags including one or more of these sequences have about the same mass (e.g., each tag has a mass that is about +/−10% from the average mass between within a specific set of tags that encode a specific variable); lack a primer-binding (e.g., constant) region; lack a constant region; or have a constant region of reduced length (e.g., a length less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 19 nucleotides, less than 18 nucleotides, less than 17 nucleotides, less than 16 nucleotides, less than 15 nucleotides, less than 14 nucleotides, less than 13 nucleotides, less than 12 nucleotides, less than 11 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, or less than 7 nucleotides).
[0204] Sequencing strategies for libraries and oligonucleotides of this length may optionally include concatenation or catenation strategies to increase read fidelity or sequencing depth, respectively. In particular, the selection of encoded libraries that lack primer-binding regions has been described in the literature for SELEX, such as described in Jarosch et al., Nucleic Acids Res. 34: e86 (2006), which is incorporated herein by reference. For example, a library member can be modified (e.g., after a selection step) to include a first adapter sequence on the 5′-terminus of the conjugate or encoded chemical entity and a second adapter sequence on the 3′-terminus of the conjugate or encoded chemical entity, where the first sequence is substantially complementary to the second sequence and result in forming a duplex. To further improve yield, two fixed dangling nucleotides (e.g., CC) are added to the 5′-terminus. In particular embodiments, the first adapter sequence is 5′-GTGCTGC-3′ (SEQ ID NO: 1), and the second adapter sequence is 5′-GCAGCACCC-3′ (SEQ ID NO: 2).
Enzymatic Ligation and Chemical Ligation Techniques
[0205] Various ligation techniques can be used to add tags to the headpiece to produce an encoded chemical entity. Accordingly, any of the binding steps described herein can include any useful ligation techniques, such as enzymatic ligation and/or chemical ligation. These binding steps can include the addition of one or more tags to the oligonucleotide headpiece of the encoded chemical entity. In particular embodiments, the ligation techniques used for any oligonucleotide provide a resultant product that can be transcribed and/or reverse transcribed to allow for decoding of the library or for template-dependent polymerization with one or more DNA or RNA polymerases.
[0206] Generally, enzymatic ligation produces an oligonucleotide having a native phosphodiester bond that can be transcribed and/or reverse transcribed. Exemplary methods of enzyme ligation are provided herein and include the use of one or more RNA or DNA ligases, such as T4 RNA ligase 1 or 2, T4 DNA ligase, CircLigase™ ssDNA ligase, CircLigase™ II ssDNA ligase, and ThermoPhage™ ssDNA ligase (Prokazyme Ltd., Reykjavik, Iceland).
[0207] Chemical ligation can also be used to produce oligonucleotides capable of being transcribed or reverse transcribed or otherwise used as a template for a template-dependent polymerase. The efficacy of a chemical ligation technique to provide oligonucleotides capable of being transcribed or reverse transcribed may need to be tested. This efficacy can be tested by any useful method, such as liquid chromatography-mass spectrometry, RT-PCR analysis, PCR analysis, electrophoresis, and/or sequencing.
Reaction Conditions to Promote Enzymatic Ligation or Chemical Ligation
[0208] The methods described herein can include one or more reaction conditions that promote enzymatic or chemical ligation between the headpiece and a tag or between two tags. These reaction conditions include using modified nucleotides within the tag, as described herein; using donor tags and acceptor tags having different lengths and varying the concentration of the tags; using different types of ligases, as well as combinations thereof (e.g., CircLigase™ DNA ligase and/or T4 RNA ligase), and varying their concentration; using poly ethylene glycols (PEGs) having different molecular weights and varying their concentration; use of non-PEG crowding agents (e.g., betaine or bovine serum albumin); varying the temperature and duration for ligation; varying the concentration of various agents, including ATP, Co(NH.sub.3).sub.6Cl.sub.3, and yeast inorganic pyrophosphate; using enzymatically or chemically phosphorylated oligonucleotide tags; using 3′-protected tags; and using preadenylated tags. These reaction conditions also include chemical ligations.
[0209] The headpiece and/or tags can include one or more modified or substituted nucleotides. In preferred embodiments, the headpiece and/or tags include one or more modified or substituted nucleotides that promote enzymatic ligation, such as 2′-O-methyl nucleotides (e.g., 2′-O-methyl guanine or 2′-O-methyl uracil), 2′-fluoro nucleotides, or any other modified nucleotides that are utilized as a substrate for ligation. Alternatively, the headpiece and/or tags are modified to include one or more chemically reactive groups to support chemical ligation (e.g. an optionally substituted alkynyl group and an optionally substituted azido group). Optionally, the tag oligonucleotides are functionalized at both termini with chemically reactive groups, and, optionally, one of these termini is protected, such that the groups may be addressed independently and side-reactions may be reduced (e.g., reduced polymerization side-reactions).
[0210] As described herein, chemical ligation which results in phosphodiester, phosphonate, or phosphorothioate linkages may be performed by reaction of a 5′- or 3′-phosphate, phosphonate, or phosphorothioate with a 5′- or 3′-hydroxyl group in the presence of cyanoimidazole and a divalent metal ion such as Zn.sup.2+.
[0211] Enzymatic ligation can include one or more ligases. Exemplary ligases include CircLigase™ ssDNA ligase (EPICENTRE Biotechnologies, Madison, Wis.), CircLigase™ II ssDNA ligase (also from EPICENTRE Biotechnologies), ThermoPhage™ ssDNA ligase (Prokazyme Ltd., Reykjavik, Iceland), T4 RNA ligase, and T4 DNA ligase. In preferred embodiments, ligation includes the use of an RNA ligase or a combination of an RNA ligase and a DNA ligase. Ligation can further include one or more soluble multivalent cations, such as Co(NH.sub.3).sub.6Cl.sub.3, in combination with one or more ligases.
[0212] Before or after the ligation step, a conjugate or encoded chemical entity can be purified. In some embodiments, the conjugate or encoded chemical entity can be purified to remove unreacted headpiece or tags that may result in cross-reactions and introduce “noise” into the encoding process. In some embodiments, the conjugate or encoded chemical entity can be purified to remove any reagents or unreacted starting material that can inhibit or lower the ligation activity of a ligase. For example, orthophosphate may result in lowered ligation activity. In certain embodiments, entities that are introduced into a chemical or ligation step may need to be removed to enable the subsequent chemical or ligation step. Methods of purifying the conjugate or encoded chemical entity are described herein. Purification of the conjugate or encoded chemical entity may be carried out by reversible immobilization of the conjugate or encoded chemical entity followed by purification and release prior to a subsequent step.
[0213] Enzymatic and chemical ligation can include poly ethylene glycol having an average molecular weight of more than 300 Daltons (e.g., more than 600 Daltons, 3,000 Daltons, 4,000 Daltons, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, or 45,000 Daltons). In particular embodiments, the polyethylene glycol has an average molecular weight from about 3,000 Daltons to 9,000 Daltons (e.g., from 3,000 Daltons to 8,000 Daltons, from 3,000 Daltons to 7,000 Daltons, from 3,000 Daltons to 6,000 Daltons, and from 3,000 Daltons to 5,000 Daltons). In preferred embodiments, the poly ethylene glycol has an average molecular weight from about 3,000 Daltons to about 6,000 Daltons (e.g., from 3,300 Daltons to 4,500 Daltons, from 3,300 Daltons to 5,000 Daltons, from 3,300 Daltons to 5,500 Daltons, from 3,300 Daltons to 6,000 Daltons, from 3,500 Daltons to 4,500 Daltons, from 3,500 Daltons to 5,000 Daltons, from 3,500 Daltons to 5,500 Daltons, and from 3,500 Daltons to 6,000 Daltons, such as 4,600 Daltons). Polyethylene glycol can be present in any useful amount, such as from about 25% (w/v) to about 35% (w/v), such as 30% (w/v).
Methods for Tagging Encoded Libraries
[0214] The methods described herein can be used to synthesize libraries having a diverse number of chemical entities that are encoded by oligonucleotide tags. The invention features methods for operatively associating oligonucleotide tags with chemical entities (e.g., compounds such as pre-existing compounds), such that encoding relationships may be established between the sequence of the tag and the identity of the chemical entity. In particular, the identity of a chemical entity can be inferred from the sequence of bases in the oligonucleotide. Using this method, a library including diverse chemical entities can be encoded with a particular set of tags.
[0215] Generally, these methods include the use of i) a chemical entity; ii) a bifunctional linker including a carbene precursor group and a cross-linking group; iii) a conjugate including an oligonucleotide headpiece and a cross-linking group; and iv) an oligonucleotide tag or unique combination of tags designed to ligate to each other. One oligonucleotide tag is bound to the oligonucleotide headpiece. Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., ligation with one or more of an RNA ligase and/or a DNA ligase) or by chemical binding (e.g., by a substitution reaction between two functional groups, such as a nucleophile and a leaving group).
[0216] This invention describes a practical method of encoding millions of individual chemical entities (e.g., pre-existing compounds) using unique combinations of encoding oligonucleotides. As an example, an encoding strategy in which each final concatenated tag set has the design Compound-Linker-Headpiece-TagA-TagB-TagC-TagD-Tailpiece can uniquely encode 6.25 million (50×50×50×50) compounds with one oligonucleotide Headpiece, 50 unique oligonucleotide TagA's, 50 unique oligonucleotide TagB's, 50 unique oligonucleotide TagC's, 50 oligonucleotide TagD's, and one oligonucleotide Tailpiece. This totals 200 unique oligonucleotide tags, one oligonucleotide Headpiece, and one oligonucleotide Tailpiece. The Headpiece and Tailpiece can contain constant primer-binding sequences or provide a functional group to allow for binding (e.g., by ligation) of a primer-binding sequence that are used for amplification and optionally are utilized for clustering and sequencing. The primer-binding sequence can be used for amplifying and/or sequencing the oligonucleotides tags of the conjugate or encoded chemical entity. Exemplary methods for amplifying and for sequencing include polymerase chain reaction (PCR), linear chain amplification (LCR), rolling circle amplification (RCA), or any other method known in the art to amplify or determine nucleic acid sequences. Dispensing well-specific combinations of these oligonucleotide tags along with the individual compounds that they will encode is readily automated.
[0217] The oligonucleotide tags may be single-stranded or double-stranded and contain orthogonal ligation overlaps that allow them to ligate in a precise spatial order even if all oligonucleotides are introduced simultaneously into a “one-pot” reaction mixture. Oligonucleotides are appropriately modified for ligation (e.g., by 5′-phosphorylation).
Methods for Screening Encoded Libraries
[0218] Next, the library can be tested and/or selected for a characteristic or function, as described herein. For example, the mixture of tagged chemical entities can be separated into at least two populations, where the first population is enriched for members that bind to a particular biological target and the second population that is less enriched (e.g., by negative selection or positive selection). The first population can then be selectively captured (e.g., by eluting from a column providing the target of interest or by incubating the aliquot with the target of interest followed by capture of the protein along with associated library members and subsequent elution of library members) and, optionally, further analyzed or tested, such as with optional washing, purification, negative selection, positive selection, or separation steps. Adaptation of these methods can yield reversible or irreversible covalent target modifiers when a library elution step is included that cleaves at least one covalent bond, either within or between the encoding tags of the library member and the matrix or within the target protein, for example using a restriction endonuclease or a protease.
[0219] Once the pre-existing compounds from the first library that bind to the target of interest have been identified, a second library of pre-existing compounds may be encoded and screened against targets of interest.
Methods for Decoding Encoded Libraries
[0220] Finally, the identity of the encoded chemical entities within a selected population can be determined by the sequence of the oligonucleotide tags. Upon correlating the sequence with encoded library members tagging history, this method can identify the individual members of the library with the selected characteristic (e.g., an increased tendency to bind to the target protein and thereby elicit a therapeutic effect). For further testing and optimization, candidate therapeutic compounds may then be prepared by synthesizing the identified library members with or without their associated oligonucleotide tags or by directly accessing individual pre-existing compounds that were used to construct the library, either with or without modification by a reactive or photoreactive linker element.
[0221] The methods described herein can include any number of optional steps to diversify the library or to interrogate the members of the library. For any tagging method described herein, successive “n” number of tags can be added with additional “n” number of ligation, separation, and/or phosphorylation steps or alternatively with “successive” ligations occurring in a “single-pot” reaction to provide a unique combinatorial catenated tag set. Exemplary optional steps include restriction of library member-associated encoding oligonucleotides using one or more restriction endonucleases; repair of the associated encoding oligonucleotides, e.g., with any repair enzyme, such as those described herein; ligation of one or more adapter sequences to one or both of the termini for library member-associated encoding oligonucleotides, e.g., such as one or more adapter sequences to provide a priming sequence for amplification and sequencing or to provide a label, such as biotin, for immobilization of the sequence; reverse-transcription or transcription, optionally followed by reverse-transcription, of the assembled tags in the conjugate or encoded chemical entity using a reverse transcriptase, transcriptase, or another template-dependent polymerase; amplification of the assembled tags in the conjugate or encoded chemical entity using, e.g., PCR; generation of clonal isolates of one or more populations of assembled tags in the conjugate or encoded chemical entity, e.g., by use of bacterial transformation, emulsion formation, dilution, surface capture techniques, etc.; amplification of clonal isolates of one or more populations of assembled tag in the conjugate or encoded chemical entity, e.g., by using clonal isolates as templates for template-dependent polymerization of nucleotides; and sequence determination of clonal isolates of one or more populations of assembled tags in the conjugate or encoded chemical entity, e.g., by using clonal isolates as templates for template-dependent polymerization with fluorescently labeled nucleotides with reversible terminator chemistry. Additional methods for amplifying and sequencing the oligonucleotide tags are described herein.
[0222] These methods can be used to identify and discover any number of chemical entities with a particular characteristic or function, e.g., in a selection step. The desired characteristic or function may be used as the basis for partitioning the library into at least two parts with the concomitant enrichment of at least one of the members or related members in the library with the desired function. In particular embodiments, the method comprises identifying a small drug-like library member that binds or inactivates a protein of therapeutic interest. In any of these instances, the oligonucleotide tags encode the chemical history of the library member and in each case a collection of chemical possibilities may be represented by any particular tag combination.
[0223] In one embodiment, the library of chemical entities, or a portion thereof, is contacted with a biological target under conditions suitable for at least one member of the library to bind to the target, followed by removal of library members that do not bind to the target, and analyzing the one or more oligonucleotide tags associated with the target. This method can optionally include amplifying the tags by methods known in the art. Exemplary biological targets include enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins involved in protein:protein interactions (e.g., ligands for receptors), receptor targets (e.g., GPCRs and RTKs), ion channels, bacteria, viruses, parasites, DNA, RNA, prions, and carbohydrates.
[0224] In another embodiment, the encoded chemical entities that bind to a target are not subjected to amplification but are analyzed directly. Exemplary methods of analysis include microarray analysis, including evanescent resonance photonic crystal analysis; bead-based methods for deconvoluting tags (e.g., by using his-tags); label-free photonic crystal biosensor analysis (e.g., a BIND® Reader from SRU Biosystems, Inc., Woburn, Mass.); or hybridization-based approaches (e.g. by using arrays of immobilized oligonucleotides complementary to sequences present in the library of tags).
[0225] In addition, chemical-reactive pairs can be readily included in solid-phase oligonucleotide synthesis schemes and will support the efficient chemical ligation of oligonucleotides. In addition, the resultant ligated oligonucleotides can act as templates for template-dependent polymerization with one or more polymerases. Accordingly, any of the binding steps described herein for tagging encoded libraries can be modified to include one or more of enzymatic ligation and/or chemical ligation techniques. Exemplary ligation techniques include enzyme ligation, such as use of one of more RNA ligases and/or DNA ligases; and chemical ligation, such as use of chemical-reactive pairs (e.g., a pair including optionally substituted alkynyl and azido functional groups).
[0226] In some embodiments, amplifying can optionally include forming a water-in-oil emulsion to create a plurality of aqueous microreactors. The reaction conditions (e.g., concentration of conjugate or encoded chemical entity and size of microreactors) can be adjusted to provide, on average, a microreactor having at least one member of a library of compounds. Each microreactor can also contain the target, a single bead capable of binding to an encoded chemical entity or a portion of the encoded chemical entity (e.g., one or more tags) and/or binding the target, and an amplification reaction solution having one or more necessary reagents to perform nucleic acid amplification. After amplifying the tag in the microreactors, the amplified copies of the tag will bind to the beads in the microreactors, and the coated beads can be identified by any useful method.
General Strategy for Tagging, Screening, and Decoding Encoded Libraries of Pre-Existing Chemical Entities
[0227] The methods described herein may involve introduction of an entire library of chemical entities (e.g., compound collection) as individual chemical entities (e.g., compounds) into each well on a one-compound, one-well basis, similar to commonly utilized processes for the generation of assay-ready plates. This may be followed by the introduction of a bifunctional linker (e.g., 3-(2-Azidoethyl)-3-methyl-3H-diazirine) at high relative concentration in an organic solvent followed by irradiation to activate the aziridine group and allow for the formation of a covalent linkage between the bifunctional linker and the compound to be encoded. Subsequent reduction of pressure may remove excess unreacted bifunctional linker and optionally all or some of the organic solvent. In the succeeding step a bifunctional headpiece oligonucleotide may be introduced into each well along with a well-specific combination of encoding tags, a ligase enzyme and a ligase-competent buffer. The encoding tags are designed to ligate to the headpiece and to each other in a precisely determined order by careful design of their ligation junctions. The headpiece also contains a strained alkyne that will react with the azide that is connected to the compound to be encoded in a copper-free click reaction since copper may interfere with the ligation efficiency or specificity.
[0228] Subsequently the contents of the individual wells may be quenched, combined and then further purified and concentrated as a mixture before ligation to a tailpiece containing a library-identifying encoding sequence along with other tag sequences as desired. Once generated aliquots of the library may be used for affinity-mediated screening either combined with other encoded libraries or not.
[0229] The one-pot ligation of a well-specific combination of tags allows for the tagging of larger libraries of pre-existing compounds (e.g., libraries of millions of compounds rather than libraries of thousands of compounds). Additionally, the present invention allows for incubating the pre-existing compounds with volatile diazirine-azide linker that upon irradiation can insert the resulting carbene into potentially multiple reactive sites on the compounds. Furthermore, this method allows for the unreacted cross-linker to be removed at low pressure, followed by conjugation of the azide to the headpiece. HTS-ready plates of libraries of pre-existing compounds are encoded with well-specific combinations of oligonucleotide tags via a single ligation.
[0230] Traditional HTS utilizes activity-based discovery of target-modulating molecules by detecting their influence upon assays with readouts derived from biochemical (e.g. enzymatic transformation of substrates), biophysical (e.g. labeled probe displacement) or biological (e.g. cell-based). Generally these assays are conducted with a low concentration of target (e.g. protein) and a high concentration of a putative target-modulating molecule (e.g., a small molecule compound that is part of a library of pre-existing compounds). Such screens are to a great extent confounded by artifacts that result from the high concentration of the small molecule such as aggregation-mediated or insolubility-mediated signal.
[0231] The opportunity to run an affinity-mediated screen on the same library of compounds but encoded by oligonucleotide tags provides an opportunity to determine which compound collection members interact with the target protein under an entirely distinct assay environment (e.g., the individual compound concentrations are low). Furthermore, solubility is conferred by the conjugated oligonucleotides, thereby offering orthogonal assay data that aids in the identification of genuine hits from the original screen. In many cases, more than half of the timeline of a project utilizing a pre-existing combinatorially-generated DNA-encoded chemical library is dedicated to the re-synthesis of off-DNA versions of the molecules enriched in the affinity-mediated library screen. Thus, the encoding of libraries of pre-existing compounds accelerates project timelines since no re-synthesis of enriched compounds identified in the screen is necessary since all compounds pre-exist within the original library or collection.
[0232] The library of pre-existing compounds may be a collection of compounds utilized by pharmaceutical companies to discover modulators of target proteins. The individual members of the collection may be aliquoted into separate compartments (e.g., individual wells of multiwall plates (e.g. 96-well plates, 384-well plates, or 1536-well plates)). Each compound within each well may be reacted by incubation with a linker, for example a volatile bifunctional linker. An example of a volatile bifunctional linker is a low molecular weight compound which includes a diazirene group (a carbene precursor) and an azide group (a cross-linking group). The diazirene functional group is reacted with the compound under suitable reaction conditions (e.g., photochemical conditions via irradiation). Irradiation activates the diazirine group, transforming it into a carbene. Photochemically activated diazirines can insert themselves into a range of covalent bonds, thereby forming covalent linkages to molecules not designed with conjugation in mind, and because they can react at multiple loci within individual molecules they can display them from multiple vectors allowing for the discovery of molecules that are inactivated by conjugation at some positions. Reduced pressure can then be used to remove the volatile unreacted bifunctional linker and the residual functionalized HTS compound can then be conjugated to an azide-reactive oligonucleotide and then encoded by the introduction of a combination of oligonucleotides that have been designed to ligate to each other and to the azide-reactive oligonucleotide in a defined order to generate an amplifiable concatenated set of oligonucleotide tags and primer-binding sequences. An example of a suitable volatile bifunctional cross-linker is 3-(2-Azidoethyl)-3-methyl-3H-diazirine.
[0233] The individual amplifiable encoded oligonucleotide-HTS deck compounds can be combined, optionally further purified and concentrated as a mixture, and subjected to affinity-mediated screens followed by polymerase-mediated amplification and sequencing to identify enriched library members. Confirmation of the target-modulating activity of individual enriched HTS deck compounds may then be established by testing individual HTS deck compounds in their off-DNA form in appropriate activity assays. There is no need to resynthesize the untagged compounds since they already exist.
EXAMPLES
Example 1—Tagging Pre-Existing Compounds
[0234] Reacting a Chemical Entity with a Bifunctional Linker Including a Carbene Precursor Group and a First Cross-Linking Group to Produce a First Conjugate
[0235] Chemical entities are sourced from libraries of pre-existing compounds and aliquoted into multiwell plates with one compound per well. These may be in solution or dry and may be placed in 96-well, 384-well or 1536-well or other spatially segregated compartments.
[0236] A bifunctional linker (e.g., a volatile bifunctional linker (VBL)) is synthesized or obtained commercially. One reactive group of the VBL can be photochemically reacted to produce a carbene. The other reactive group is an azide cross-linking group suitable for click chemistry. An example of a VBL is:
##STR00008##
The synthesis of linker 1 (3-(2-Azidoethyl)-3-methyl-3H-diazirine) is reported in Liang et al., Angew. Chem. Int. Ed. Engl. 56 (10):2744-2748 (2017).
[0237] Then linker 1 and dimethylsulfoxide (DMSO) are added to each well of the succession of multiwell plates, or other spatially addressable compartments, and irradiated at 365 nm for 30 minutes. The resulting first conjugate in each well is purified by removing unreacted linker 1 by evaporation under reduced pressure (e.g., about 400 torr) and elevated temperature (e.g., about 25-30° C.).
[0238] Each first conjugate has the structure of the following:
##STR00009##
where CE represents a structure including a chemical entity.
[0239] Synthesis of a Second Conjugate Including an Oligonucleotide Headpiece and a Cross-Linking Group
[0240] A second conjugate including an oligonucleotide headpiece and a cross-linking group is synthesized from a primary amine-terminated oligonucleotide headpiece and a linker including a dibenzocyclooctyne-amino (DBCO) group. An example of an amine-terminated oligonucleotide headpiece is headpiece 1 (SEQ ID NO: 3), which has the following structure:
##STR00010##
An example of a linker including a DBCO group is linker 2, which has the following structure:
##STR00011##
[0241] Headpiece 1 and linker 2 are reacted together so that the NHS ester group of linker 2 reacts with the amine group of headpiece 1 to generate a conjugate (conjugate 2; SEQ ID NO: 4), which includes an oligonucleotide headpiece and a DBCO cross-linking group:
##STR00012##
Conjugate 2 is purified using HPLC.
[0242] Reacting the First Conjugate with a Second Conjugate to Produce a Third Conjugate
[0243] To each well is then added conjugate 2 in aqueous buffer, and the resulting mixture is incubated to allow reaction (e.g., click chemistry) between the azide of conjugate 1 and the strained alkyne of conjugate 2 to produce conjugate 3 (SEQ ID NO: 5), which has the following structure:
##STR00013##
A simplified illustration of conjugate 3, as used herein, is shown below:
##STR00014##
[0244] Ligating Oligonucleotide Tags to the Oligonucleotide Headpiece of the Third Conjugate
[0245] Subsequently an aqueous solution of a well-specific, and therefore compound-specific, collection of DNA tags is added that are designed to permit only one order of ligation by careful design of orthogonal overlap architectures.
[0246] An example of an encoding strategy is illustrated below:
##STR00015##
In this example compound collections are encoded using a four-register tag system in which compounds (e.g., pre-existing compounds) are presented in plates and Tag A encodes the identity of the row of each plate, Tag B encodes the identity of the column of each plate, Tag C encodes the identity of each plate, and variation at Tag D allows for the preceding tags to be subsequently reused in a different context. If 400 tags are available in total, divided equally between each register, then a total of 100 million compounds may each be uniquely encoded.
[0247] After the ligation incubation is complete then the contents of each well are combined and quenched, e.g., with EDTA, and the individual encoded compounds that comprise the entire library are thereby pooled together. The library is concentrated by precipitation and purified by HPLC. The library is then closed by a further ligation of a closing tag or tailpiece that introduces a library identifying sequence and a constant sequence for primer-binding during amplification and may optionally contain other tags and/or sequences helpful for downstream operations including clustering and sequencing.
[0248] Screening of Encoded Libraries
[0249] This library is then used to discover individual members that are able to bind to protein or other targets of interest by incubating with the target of interest, capture of the target, washing away of non-binding library members and the elution of the protein-associated members either by protein denaturation, tag cleavage or specific elution. The encoding DNA of the output population is then amplified and sequenced and compared with a corresponding sample derived from the input population to identify compounds that are enriched in the output. Compounds of interest are then sourced from the pre-existing collection and tested in target modulation assays to determine which may be considered hits.
Example 2—Synthesis of a Conjugate DBCO-HP006 that Includes an Oligonucleotide Headpiece and a Cross-Linking Group
[0250] Headpiece HP006, chemically phosphorylated at its 5′ end, whose sequence is (p)CCTGTGTTZTTCACAGGCCT (SEQ ID NO: 6), where Z stands for the mdC(TEG-Amino) modification, was utilized.
[0251] To 300 μL of a solution of HP006 (10 mM) in water was added 8 μL of water, 25 μL of Pierce 1 M Borate Buffer pH 8.5 (Thermo Fisher), and 167 uL of a solution of DBCO-PEG4-NHS ester (BroadPharm) (30 mM) in DMSO. The mixture was allowed to stand at room temperature for 2 days. To a 62-μL fraction of this mixture was added ethanol (560 μL), and the precipitate was collected after centrifugation. The precipitate was washed with 80% ethanol (650 μL). The washed precipitate was allowed to dry by exposure to air, and then reconstituted in water (125 μL). The concentration of the product DBCO-HP006 was determined on a nanodrop UV spectrophotometer to be 2.7 mM (90%).
[0252] LCMS of the product DBCO-HP006 is shown in
Example 3—Conjugating Pre-Existing Compounds to Linker 1 and DBCO-HP006
[0253] To the bottom of each well of a 96-well natural-colored polypropylene PCR plate was added a DMSO solution containing 18 mM or 6 mM of a pre-existing compound and 200 mM of Linker 1. The plate was irradiated on an Alpha Innotech AIML-26 Transilluminator at 365 nm (6×8 W) for 10 minutes. A 2 μL fraction of each reaction mixture was then mixed into 20 μL of 25 μM DBCO-HP006 in 1×T4 DNA ligase buffer (made from 10× ligase buffer from Thermo Fisher) and allowed to stand at room temperature overnight.
[0254] Twelve pre-existing compounds were subjected to this conjugation procedure (Table 1). The crude conjugation mixtures were analyzed by LCMS. Conjugation products with the expected m/z values could be detected for ten out of the twelve starting compounds. The results are summarized in Table 1. LCMS data are shown in
TABLE-US-00001 TABLE 1 Summary of LCMS analysis results for conjugating pre-existing compounds to Linker 1 and DBCO-HP006 Calculated m/z for [M − 8H].sup.8− of compound/ linker 1/DBCO- Pre-existing compound HP006 conjugate Observed m/z Tamoxifen 915.9 915.8 (CAS# 10540-29-1) (FIG. 2A and FIG. 2B) Elacestrant, RAD1901 926.8 926.7 (CAS# 722533-56-4) (FIG. 3A and FIG. 3B) AZD 9496 924.8 Expected product was (CAS# 1639042-08-2) not observed. Bazedoxifene 928.3 928.1 (CAS# 198481-32-2) (FIG. 4A and FIG. 4B) 17β-Estradiol 903.5 903.4 (CAS# 50-28-2) (FIG. 5A and FIG. 5B) (Z)-4-hydroxy Tamoxifen 917.9 917.8 (CAS# 68047-06-3) (FIG. 6A and FIG. 6B) Fulvestrant 945.3 Expected product was (CAS# 129453-61-8) not observed. PPT 917.8 917.6 (CAS# 263717-53-9) (FIG. 7A and FIG. 7B) MPP 928.2 928.1 (CAS# 911295-24-4) (FIG. 8A and FIG. 8B) WAY 200070 907.8 907.7 (CAS# 440122-66-7) (FIG. 9A and FIG. 9B) Estriol 905.5 905.4 (CAS# 50-27-1) (FIG. 10A and FIG. 10B) DPN 899.4 899.3 (CAS# 1428-67-7) (FIG. 11A and FIG. 11B)
Example 4—One-Pot Ligation of an Oligonucleotide Headpiece, a Headpiece Extension, and Four Tags that May be Used to Encode Compound Identity
[0255] Two DNA oligonucleotides, chemically phosphorylated at their respective 5′ ends, whose sequences are (p)TGGCTATCCTGGCTGAGG (SEQ ID NO: 7) and (p)CAGCCAGGATAG (SEQ ID NO: 8), were combined in equimolar ratio to make a 1 mM solution of double-stranded EXT00001.
[0256] Two DNA oligonucleotides, chemically phosphorylated at their respective 5′ ends, whose sequences are (p)CCAAAGAGTGGAGCTAAG (SEQ ID NO: 9) and (p)AGCTCCACTCTT (SEQ ID NO: 10), were used. as a pre-mixed 1 mM solution of double-stranded TagA.
[0257] Two DNA oligonucleotides, chemically phosphorylated at their respective 5′ ends, whose sequences are (p)GCTATGGAGCCACTACTT (SEQ ID NO: 11) and (p)TAGTGGCTCCAT (SEQ ID NO: 12), were used as a pre-mixed 1 mM solution of double-stranded TagB.
[0258] Two DNA oligonucleotides, chemically phosphorylated at their respective 5′ ends, whose sequences are (p)AGCGGATCTAGCCAATGC (SEQ ID NO: 13) and (p)TTGGCTAGATCC (SEQ ID NO: 14), were used as a pre-mixed 1 mM solution of double-stranded TagC.
[0259] Two DNA oligonucleotides, chemically phosphorylated at their respective 5′ ends, whose sequences are (p)CATACATACGCGACTGCA (SEQ ID NO: 15) and (p)AGTCGCGTATGT (SEQ ID NO: 16), were used as a pre-mixed 1 mM solution of double-stranded TagD.
[0260] To a 50 μL mixture of six duplexed oligonucleotide components (final concentrations: 20 μM HP006, 1.05 molar equivalent of EXT00001, 1.1 molar equivalent of TagA, 1.15 molar equivalent of TagB, 1.2 molar equivalent of TagC, 1.25 molar equivalent of TagD) in 1× ligase buffer (using 10× ligase buffer from Thermo Fisher) was added 1.5 ul of T4 DNA ligase (Thermo Fisher). Six negative control reactions were set up with the same procedure except that, in each negative control reaction, one of the duplexed oligonucleotide components was replaced by an equivalent volume of water. The reactions were incubated at 16° C. in a thermocycler for 2 days. The reaction mixtures were analyzed by electrophoresis on a 4% E-Gel high-resolution agarose gel containing ethidium bromide. The gel image is shown in
[0261] The one-pot ligation reaction produced one major DNA ligation product that is longer than the major products produced by all the negative control reactions, proving that the one-pot ligation reaction ligated all the oligonucleotide components in a defined sequence.
OTHER EMBODIMENTS
[0262] All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.
[0263] Various modifications and variations of the described method and system will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention.